Culture and Identification of Umbilical Cord Mesenchymal Stem cells

Sterilization of Fresh Umbilical Cord(1) The fresh umbilical cord is immediately brought to the cell culture room for aseptic separation. A DPBS-double antibiotic mixture is prepared by adding 1% penicillin-streptomycin to DPBS buffer to prevent contamination.(2) Three 250 mL glass beakers, which have been washed and autoclaved, are prepared. 100 mL of DPBS-double antibiotic mixture is added to each beaker. The umbilical cord is placed in beaker 1 for soaking and cleaning, and medical tweezers are used to try to squeeze out the blood in the umbilical cord vessels.(3) The soaked umbilical cord is then placed in a 10 cm diameter cell culture dish, and carefully wiped with gauze soaked in iodine.(4) After wiping with iodine, the umbilical cord is placed in beaker 2 and cut into segments of 3-4 cm. A 5 mL sterile syringe is used to flush out the blood in the umbilical cord vessels with DPBS-double antibiotic mixture.Acquisition of Wharton's Jelly(1) The umbilical cord segments are clamped into a 6 cm diameter cell culture dish using tweezers. A 14# medical toothed tweezer and a 10# medical ophthalmic tweezer are used to perform blunt dissection of the umbilical cord, carefully dissecting the two umbilical veins, one umbilical artery, and the umbilical cord epidermis.(2) The purified Wharton's jelly obtained from dissection is torn into strips and placed in beaker 3. A pipette is used to aspirate DPBS-double antibiotic mixture to rinse and soak the Wharton's jelly twice.(3) The Wharton's jelly is then placed in a 10 cm diameter cell culture dish and gently cut into a uniform fine paste of approximately 2 mm tissue pieces using scissors. The tissue pieces are carefully and evenly spread out in the dish using tweezers, with each dish containing 70% of the Wharton's jelly tissue pieces. The dishes are placed in a 37°C CO2 incubator for 30 minutes to promote adherence of the tissue pieces.(4) To prepare the tissue piece culture medium, 500 mL of DMEM low-glucose basic medium is mixed with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, and 1% L-glutamine. After mixing, 10 mL of medium is gently added along the edge of each dish.(5) Half of the medium is replaced after 3 days of static culture, and then the medium is changed every 3 days to observe whether cells have migrated out along the edges of the tissue pieces.Identification of Surface Markers of Umbilical Cord-Derived Mesenchymal Stem Cells by Flow Cytometry Umbilical cord-derived mesenchymal stem cells (UC-MSCs) of the third passage with good growth were selected and cultured until they reached a confluence of 90%-99%. The cells were then digested with stem cell digestive solution for 3 minutes and collected into centrifuge tubes. After centrifugation at 1000 rpm for 5 minutes, the supernatant was discarded, and the cell precipitate was resuspended in PBS. The cells were then counted and adjusted to a concentration of 5x10^6/mL.Seven EP tubes were labeled and numbered. Antibodies were added to tubes 1-4, specifically CD90-FITC, CD44-PE, CD105-PerCP-Cy5.5, and CD73-APC, respectively. Tube 5 served as a blank control. Tube 6 contained a mixture of positive antibody isotype controls (mIgG1-FITC, mIgG1-PerCP-Cy5.5, mIgG1-APC) and negative antibody isotype controls (mIgG1-PE, mIgG2a-PE). Tube 7 contained a mixture of positive antibodies (CD90-FITC, CD105-PerCP-Cy5.5, CD73-APC) and a mixture of negative antibodies (CD34-PE, CD11b-PE, CD19-PE, CD45-PE, HLA-DR-PE). 100 μL of cell suspension was added to each tube, and the EP tubes were incubated on ice in the dark for 30 minutes.After incubation, 300 μL of PBS was added to each tube, and the cells were centrifuged at 4°C and 1000 rpm for 5 minutes. This was repeated twice. After the second centrifugation, 300 μL of PBS was added to resuspend the cells, and the cells were filtered through a mesh into flow cytometry tubes that were already labeled with the corresponding numbers. The tubes were kept on ice during transportation to the flow cytometry machine. Flow cytometry was performed, and the cell phenotypes were analyzed using FlowJo software. Identification of Cell Induction and Differentiation Adipogenic Induction and DifferentiationCells in the logarithmic growth phase were seeded into 6 cm diameter dishes at a density of 2x102 and cultured at 37°C in a 5% CO2 environment until they reached a confluence of 90-100%. The supernatant was discarded, and adipogenic induction medium was added. The cells were cultured for approximately 3 days in this medium, then switched to adipogenic maintenance medium for 1 day, followed by another 3 days in adipogenic induction medium. This induction cycle was repeated for 21 days, with media changes at the specified intervals, and cell morphology was observed. The timing of terminating the induction was determined based on the number and size of lipid droplets formed in the cells, followed by staining and identification. The medium was aspirated, and the cells were washed once with an appropriate amount of 1xPBS. After discarding the wash, an appropriate amount of 4% neutral formalin solution was added to cover the bottom of the culture dish and fixed at room temperature for 30-60 minutes. The fixative was discarded, and the cells were washed twice with 1xPBS. An oil red O working solution was prepared by mixing oil red O stock solution with physiological saline (3:2 ratio). The working solution could be centrifuged to precipitate any supersaturated precipitates. An appropriate amount of oil red O working solution was added to the cleaned induction wells and stained for 30 minutes. The staining solution was aspirated, and the cells were washed twice with 1xPBS and then covered with an appropriate amount of 1xPBS to prevent drying. The adipogenic staining effect was observed under a microscope, and images were captured and assessed for induction success. Osteogenic Induction and DifferentiationCells in the logarithmic growth phase were seeded into coated culture dishes at a density of 2.0x102 and cultured at 37°C in a 5% CO2 environment until they reached a confluence of 60-70%. The supernatant was discarded, and osteogenic induction medium was added. The cells were cultured for approximately 21 days in this medium, with media changes every 2 days, and cell morphology was observed. The timing of terminating the induction was determined based on the formation of calcium salt crystals and calcified nodules in the cells, followed by staining and identification. The medium was aspirated, and the cells were washed once with an appropriate amount of 1xPBS. After discarding the wash, an appropriate amount of 4% neutral formalin solution was added to cover the bottom of the culture dish and fixed at room temperature for 30-60 minutes. The fixative was discarded, and the cells were washed twice with 1xPBS. An appropriate amount of alizarin red staining solution was added for 3-5 minutes, then aspirated, and the cells were washed twice with 1xPBS and covered with an appropriate amount of 1xPBS to prevent drying. The osteogenic staining effect was observed under a microscope, and images were captured and assessed for induction success. When the induction was successful, calcified nodules bound to alizarin red dye appeared red or orange. Chondrogenic Induction and DifferentiationCells in the logarithmic growth phase were digested, counted, and resuspended in chondrogenic induction medium. After centrifugation, the cell density was adjusted to 1.0-2.0x105 cells) was added dropwise to the center of a 24-well plate. The plate was incubated at 37°C in a 5% CO2 environment for 2-3 hours to allow the cells to adhere. After 2-3 hours, 1 mL of chondrogenic induction medium was added to each well for normal culture. The medium was changed every 2-3 days, and this induction cycle was repeated for 28 days. The medium was aspirated, and the cells were washed once with an appropriate amount of 1xPBS. After discarding the wash, an appropriate amount of 4% neutral formalin solution was added to cover the bottom of the culture dish and fixed at room temperature for 30-60 minutes. The fixative was discarded, and the cells were washed twice with 1xPBS. An appropriate amount of alcian blue staining solution was added to the cleaned culture dish and stained in the dark for 30 minutes. The staining solution was aspirated, and the cells were washed twice with 1xPBS and then covered with an appropriate amount of 1xPBS to prevent drying. The chondrogenic staining effect was observed under a microscope, and images were captured and assessed for induction success. When the induction was successful, the acidic mucopolysaccharides in the cartilage tissue were stained blue-green by alcian blue.